PROJECT SUMMARY/ABSTRACT The major goal of HIV-1 vaccine development is the design of immunogens capable of inducing broadly neutralizing antibodies (bnAbs) against circulating viruses. Although a number of bnAbs have been identified in HIV-infected individuals who developed broadly neutralizing responses over time, eliciting these bnAbs by immunization is unsuccessful thus far. Because it is highly conserved, CD4 binding site (CD4BS) on HIV-1 envelope glycoprotein (Env) is considered a main target for bnAbs, and the anti-CD4BS VRC01-class bnAbs are of particular interest because of their exceptional neutralization potency and breadth. Recent studies showed that the predicted germline precursors of VRC01-class bnAbs lack detectable affinity for native HIV-1 Env. This may explain the failure of all Env immunogens to induce anti-CD4BS bnAb responses. An engineered gl-targeting gp120 outer domain immunogen (eOD-GT) that contains multiple mutations including the removal of key glycosylations at positions 276 and 463 has been reported. Studies showed that eOD-GT6 bound to gl-VRC01, and activates nave B cells expressing gl-bnAbs in human Ig knock-in mice, but did not elicit bnAbs, while in contrast native-like Env trimers failed to activate the nave B cells but were needed to select for bnAbs. These data suggest that vaccination to elicit bnAbs may require immunization with a succession of related immunogens, or with a native-like immunogen that binds to bnAb germline precursor. Studies on the glycan profiles showed that there were vast differences in the glycosylation of Env produced in different systems. We therefore produced gp140 trimers in HIV-1 natural host T-lymphocytes to mimic the natural post-translational modification. Our preliminary data showed that the gp140 ? only when produced in T- lymphcytes, not in 293T cells ? was able to bind gl-VRC01. The gp140 produced in T-lymphocytes also exhibits significantly increased binding affinities to other bnAbs, including glycan-dependent PGT126 bnAb and anti-MPER 10E8 bnAb, In this proposal, we will evaluate immunogenicity of the gp140 immunogens produced in T-lymphocytes. The gp140 produced in T-lymphocytes is the first intact, native-like Env trimer in the HIV/AIDS vaccine field that binds the germline precursor of VRC01 bnAb. It binds gl-VRC01 with an affinity similar to eOD-GT6, therefore, like eOD-GT6, it should be able to activate nave B cells expressing gl-VRC01 in human Ig knock-in mice, and as an intact Env trimer, it should also be able to drive the Ab maturation to finally elicit VRC01-like bnAbs. Determining whether an intact, native-like Env immunogen that binds the germline precursor of VRC01 bnAb can elicit VRC01-like antibodies will have a considerable impact on HIV vaccine design.